Method of using a novel phosphodiesterase in pharmaceutical screeing to identify compounds for treatment of neoplasia

ABSTRACT

A method for identifying compounds useful for the treatment of neoplasia involves acertaining whether such compounds exhibit an ability to inhibit a PDE that is characterized by cGMP specificity, cooperative kinetic behavior and insensitivity to phosphorylation.

BACKGROUND OF THE INVENTION

This invention relates to a novel cGMP-specific phosphodiesterase thatprovides a method for identifying compounds potentially useful for thetreatment and prevention of pre-cancerous and cancerous lesions inmammals.

Cancer and precancer research is replete with publications that describevarious biochemical molecules that are over-expressed in neoplastictissue, leading one research group after another to research whetherspecfic over-expressed molecules are responsible for the disease, andwhether, if the over-expression were inhibited, neoplasia could bealleviated. For example, in familial adenomatous polyposis (“FAP”),Waddell in 1983 (Waddell, W. R. et al., “Sulindac for Polyposis of theColon,” Journal of Surgical Oncology, 24:83-87, 1983) hypothesized thatsince prostaglandins were over-expressed in such polyps, non-steroidalanti-inflammatory drugs (“NSAIDs”) should alleviate the conditionbecause NSAIDs inhibited prostaglandin synthetase (PGE₂) activity. Thus,he administered the nonsteroidal anti-inflammatory drug (“NSAID”)sulindac (an inhibitor of PGE₂) to several FAP patients. Waddeldiscovered that polyps regressed and did not recur upon such therapy.PGE₂ inhibition results from the inhibition of cyclooxygenase (COX)caused by NSAIDs. The success by Waddell and the PGE₂/COX relationshipseemingly confirmed the role of two other biochemical targets—PGE₂ andCOX—in carcinogenesis, and the subsequent literature reinforced theseviews.

Sulindac and other NSAIDs when chronically administered, aggravate thedigestive tract where PGE₂ plays a protective role. In addition, theyexhibit side effects involving the kidney and interference with normalblood clotting. Thus for neoplasia patients, such drugs are not apractical chronic treatment for FAP. These side effects also prohibitNSAIDs' use for any other neoplasia indication requiring long-term drugadministration.

Recent discoveries have lead scientists away from the COX/PEG₂ targets,since those targets do not appear to be the primary (or perhaps evensecondary targets) to treat neoplasia patients successfully. Pamukcu etal., in U.S. Pat. No. 5,401,774, disclosed that sulfonyl compounds, thathave been reported to be practically devoid of PGE2 and COX inhibition(and therefore not NSAIDs or anti-inflammatory compounds) unexpectedlyinhibited the growth of a variety of neoplastic cells, including colonpolyp cells. These sulfonyl derivatives have proven effective in ratmodels of colon carcinogenesis, and one variant (now referred to asexisulind) has proven effective in human clinical trials with FAPpatients.

Thus like so many other proteins over-expressed in neoplasias, PGE₂/COXover-expression is not a cause of neoplasia, rather a consequence. Butthe discoveries by Pamucku et al., however, have raised the questionabout how their compounds act —what do such compounds do to neoplasticcells?

Piazza, et al. (in U.S. Pat. No. 5,858,694) discovered that compounds(such as the sulfonyl compounds above) inhibited cyclic GMP,phosphodiesterase, namely, PDE5 and that other such compounds could bescreened using that enzyme, which could lead to the discovery of stillother pharmaceutical compositions that are anti-neoplastic, and that canbe practically devoid of COX or PGE2 inhibition. In addition,anti-neoplastic PDE5-inhibiting compounds can induce apoptosis (a formof programmed cell death or suicide) in neoplastic cells, but not innormal cells. Thus, the conventional wisdom that chemotherapeuticscannot be effective without also killing normal cells is being reversedby such discoveries.

Further, new research presented below has shown that not all compoundsexhibiting PDE5 inhibitions induce apoptosis in neoplastic cells. Forexample, the well-known PDE5 inhibitors, zaprinast and sildenafil, donot induce apoptosis, or even inhibit cell growth in neoplastic cells inour hands, as explained below. However, because pro-apoptotic PDE5inhibitors induced apoptosis selectively (i.e., in neoplastic but not innormal cells), and many did so without substantial COX inhibition, theusefulness of PDE5 as a screening tool for desirable anti-neoplasticcompounds is unquestioned.

However, an even more accurate and selective screening tool than PDE5 tofind anti-neoplastic, pro-apoptotic but safe compounds is desirable.

SUMMARY OF THE INVENTION

In the course of researching why some PDE5 inhibitors induced apoptosiswhile others did not, we uncovered a form of cyclic GMP-specificphosphodiesterase, not previously described. This new phosphodiesteraseactivity was previously uncharacterized, possibly because it isexpressed only in neoplastic tissue, or perhaps because it is a mutationof a known/characterized PDE. This new PDE is useful in screeningpharmaceutical compounds for desirable anti-neoplastic properties.

In its broadest aspects, this new PDE is characterized by having:

(a) cGMP specificity over cAMP

(b) positive cooperative kinetic behavior in the presence of cGMPsubstrate;

(c) submicromolar affinity for cGMP; and

(d) insensitivity to incubation with purified cGMP-dependent proteinkinase

Other characteristics of this novel PDE include: it has a reducedsensitivity to inhibition by zaprinast and E4021, it can be separatedfrom classical PDE5 activity by anion-exchange chromatography, it is notactivated by calcium/calmodulin, and it is insensitive to rolipram,vinpocetine and indolodan.

This invention relates to a novel in vitro method for screening testcompounds for their ability to treat and prevent neoplasia, especiallypre-cancerous lesions, safely. In particular, the present inventionprovides a method for identifying test compounds that can be used totreat and prevent neoplasia, including precancerous lesions. Thecompounds so identified can have minimal side effects attributable toCOX inhibition and other non-specific interactions. The compounds ofinterest can be tested by exposing the novel PDE described above to thecompounds, and if a compound inhibits this novel PDE, the compound isthen further evaluated for its anti-neoplastic properties.

One aspect of this invention, therefore, involves a screening method toidentify a compound effective for treating neoplasia that includesascertaining the compound's inhibition of this novel PDE and itsinhibition of COX. Preferably, the screening method of this inventionfurther includes determining whether the compound inhibits the growth oftumor cells in a cell culture.

In another aspect, the screening method of this invention involvesdetermining the COX inhibition activity of the compound, determining theinhibition activity of the compound against this novel PDE, anddetermining whether the compound induces apoptosis in tumor cells.

By screening compounds in this fashion, potentially beneficial andimproved compounds for treating neoplasia can be identified more rapidlyand with greater precision than possible in the past. Further benefitswill be apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from SW480 neoplastic cells, as assayed from a the eluent froma DEAE-Trisacryl M column;

FIG. 2 is a graph of cGMP activities of the reloaded cGMPphosphodiesterases obtained from SW480 neoplastic cells, as assayed froma the eluent from a DEAE-Trisacryl M column;

FIG. 3 is a graph of the kinetic behavior of the novel PDE of thisinvention;

FIG. 4 illustrates the effect of the sulfide derivative of sulindac andthe sulfone derivative of sulindac (a.k.a. exisulind) on purifiedcyclooxygenase activity.

FIG. 5 illustrates the effects of test compounds B and E on COXinhibition.

FIG. 6 illustrates the inhibitory effects of sulindac sulfide andexisulind on PDE4 and PDE5 purified from cultured tumor cells.

FIG. 7A illustrates the effects of sulindac sulfide on cGMP levels inHT29 cells.

FIG. 7B illustraes the effects of sulindac sulfide on cAMP levels inHT29 cells.

FIG. 8 illustrates the phosphodiesterase inhibitory activity of compoundB.

FIG. 9 illustrates the phosphodiesterase inhibitory activity of compoundE.

FIG. 10 illustrates the effects of sulindac sulfide and exisulind onapoptosis and necrosis of HT-29 cells.

FIG. 11 illustrates the effects of sulindac sulfide and exisulind onHT-29 cell growth inhibition and apoptosis induction as determined byDNA fragmentation.

FIG. 12 illustrates the apoptosis inducing properties of compound E.

FIG. 13 illustrates the apoptosis inducing properties of compound B.

FIG. 14 illustrates the effects of sulindac sulfide and exisulind ontumor cell growth.

FIG. 15 illustrates the growth inhibitory and apoptosis-inducingactivity of sulindac sulfide and control (DMSO).

FIG. 16 illustrates the growth inhibitory activity of compound E.

FIG. 17 illustrates the inhibition of pre-malignant, neoplastic lesionsin mouse mammary gland organ culture by sulindac metabolites.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. The NovelcGMP-Specific Phosphodiesterase

A. Its Isolation

The isolated cGMP-specific phosphodiesterase was prepared from the humancarcinoma cell line commonly referred to as SW480 available from theAmerican Tissue Type Collection in Rockville, Md., U.S.A. SW480 is ahuman colon cancer cell line that originated from moderatelydifferentiated epithelial adenocarcinoma.

To isolate the novel phosphodiesterase, approximately four hundredmillion SW480 cells at confluence were scraped from 150 cm² tissueculture dishes after two washes with 10 ml cold PBS and pelleted bycentrifugation. The cells were resuspended in homogenization buffer (20ml TMPI-EDTA-Triton pH 7.4:20 mM Tris-HOAc, 5 mM MgAc₂, 0.1 mM EDTA,0.8% Triton-100, 10 μM Benzamidine, 10 μM TLCK, 2000 U/mL Aprotinin, 2μM Leupeptin, 2 μM Pepstatin A) and homogenized on an ice bath using apolytron tissumizer ( three times, 20 seconds/pulse). The homogenizedmaterial was centrifuged at 105,000 g for 60 minutes at 4° C. in aBeckman L8 ultracentrifuge, and the supernatant was diluted withTMPI-EDTA (60 ml) and applied to a 10-milliliter DEAE-Trisacryl M columnpre-equilibrated with TMPI-EDTA buffer. The loaded column was washedwith 60 mL of TM-EDTA, and PDE activities were eluted with a 120 mllinear gradient of NaOAc (0-0.5 M) in TM-EDTA, at a flow rate of 0.95ml/minute, 1.4 ml/fraction. Eighty fractions were collected and assayedfor cGMP hydrolysis immediately (i.e. within minutes). FIG. 1. shows thecolumn's elution profile, revealing two initial peaks of cGMP PDEactivity, A and B, which were eluted by 40-50 mM and 70-80 mM NaOAC.Respectively. As explained below, peak A is PDE5, whereas peak B is thenovel phosphodiesterase of this invention.

Cyclic nucleotide PDE activity of each fraction was determined using themodified two-step radioisotopic method of Thompson et al. (Thompson W J,et al, Adv Cyclic Nucleotide Res 10: 69-92, 1979), as further describedbelow. The reaction was in 400 μl containing Tris-HCl (40 mM; pH 8.0),MgCl₂(5 mM), 2-mercaptoethanol (4 mM), bovine serum albumin (30 μg),cGMP (0.25 μM-5 μM) with constant tritiated substrate (200,000 cpm). Theincubation time was adjusted to give less than 15% hydrolysis. Themixture was incubated at 30° C. followed by boiling for 45 seconds tostop the reaction. Then the mixture was cooled, snake venom (50 μg)added, and the mixture was incubated at 30° C. for 10 minutes. MeOH (1ml) was added to stop the reaction, and the mixture was transferred toan anion-exchange column (Dowex 1-X8, 0.25 ml resin). The eluant wascombined with a second ml of MeOH, applied to the resin, and afteradding 6 ml scintillation fluid tritium activity was measured using aBeckman LS 6500 for one minute.

To fractionate cGMP hydrolytic activity of peaks A and B further,fractions 15 to 30 of the original 80 were reloaded onto theDEAE-Trisacryl M column and eluted with a linear gradient of NaOAc(0-0.5 M) in TM-EDTA. Fractions were again immediately assayed for cGMPhydrolysis (using the procedure described above with 0.2, 2, 5 μMsubstrate), the results of which are graphically presented in FIG. 2.One novel observation about peak B illustrated in FIG. 2 is thatincreasing substrate concentration of cGMP dramatically enhancedactivity when contrasted to peak A. Peak A activity shows apparentsubstrate saturation of high affinity catalytic sites.

B. cGMP-Specifilty of PDE Peaks A and B

Each fraction from the DEAE column was also assayed for cGMP-hydrolysisactivity (0.25 μM cGMP) in the presence or absence of Ca⁺⁺, or Ca⁺⁺-CaMand/or EGTA and for cAMP (0.25 μM cAMP) hydrolysis activity in thepresence or absence of 5 μM cGMP. Neither PDE peak A and peak B(fractions 5-22; see FIG. 1) hydrolyzed cAMP significantly, establishingthat neither was a cAMP hydrolysing gene family of PDE (i.e. a PDE 1, 2,3).

Ca++ (with or without calmodulin) failed to activate either cAMP or cGMPhydrolysis activity of either peak A or B, and cGMP failed to activateor inhibit cAMP hydrolysis. Such results establish that peaks A and Bconstitute cGMP-specific PDEs but not PDE1, PDE2, PDE3 or PDE4.

For PDE peak B, as discussed below, cyclic GMP activated the cGMPhydrolytic activity of the enzyme, but did not activate any cAMPhydrolytic activity. This reveals that PDE peak B—the novelphosphodiesterase of this invention—is not a cGMP-stimulated cAMPhydrolysis (“cGS”) or among the PDE2 family isoforns because the knownisoforms of PDE2 hydrolyze both cGMP and cAMP.

C. Peak A Is A PDE5, But Peak B—A New cGMP-Specific PDE—Is Not

To characterize any PDE isoform, kinetic behavior and substratepreference should be assessed.

Peak A showed typical “PDE5” characteristics. For example, the K_(m) ofthe enzyme for cGMP was 1.07 μM, and Vmax was 0.16 nmol/min/mg. Inaddition, as discussed below, zaprinast (IC₅₀=1.37 μM) and E4021 (IC₅₀=3nM) and sildenafil inhibited activity of peak A. Further, zaprinastshowed inhibition for cGMP hydrolysis activity of peak A, consistentwith results reported in the literature.

PDE Peak B showed considerably different kinetic properties as comparedto PDE peak A. For example, in Eadie-Hofstee plots of Peak A, cyclic GMPhydrolysis shows single line with negative slope with increasingsubstrate concentrations, indicative of Michaelis-Menten kineticbehavior. Peak B, however, shows the novel property for cGMP hydrolysisin the absence of cAMP of a decreasing (apparent K_(m)=8.4), thenincreasing slope (K_(m)<1) of Eadie-Hotfstee plots with increasing cGMPsubstrate (see, FIG. 3). Thus, this establishes Peak B's submicromolaraffinity for cGMP (i.e., where K_(m)<1).

Consistent with the kinetic studies (i.e. FIG. 3) andpostive-cooperative kinetic behavior in the presence of cGMP substrate,was the increased cGMP hydrolytic activity in the presence of increasingconcentrations of cGMP substrate. This was discovered by comparing 0.25μM, 2 μM and 5 μM concentrations of cGMP in the presence of PDE peak Bafter a second DEAE separation to rule out cAMP hydrolysis and to ruleout this new enzyme being a “classic” PDE5. Higher cGMP concentrationsevoked disproportionately greater cGMP hydrolysis with PDE peak B, asshown in FIG. 2.

These observations suggest that cGMP binding to the peak B enzyme causesa conformational change in the enzyme.

D. Zaprinast- and Sildenafil-Insensitivity of PDE Peak B Relative toPeak A, and Their Effects on Other PDE Inhibitors

Different PDE inhibitors were studied using twelve concentrations ofdrug from 0.01 to 100 μM and substrate concentration of 0.25 μM ³H-cGMP.IC₅₀ values were calculated with variable slope, sigmoidal curve fitsusing Prism 2.01 (GraphPad). The results are shown in Table 1. Whilecompounds E4021 and zaprinast inhibited peak A, (with high affinities)IC₅₀ values calculated against peak B are significantly increased (>50fold). This confirms that peak A is a PDE5. These data furtherilllustrate that the novel PDE of this invention is, for all practicalpurposes, zaprinast-insensitive and E4021-insensitive.

TABLE 1 Comparison of PDE Inhibitors Against Peak A and Peak B (cGMPHydrolysis) Ratio (IC₅₀ PDE Family IC₅₀ IC₅₀ Peak A/ Compound InhibitorPeak A (μM) Peak B (μM) Peak B) E4021 5 0.003 8.4 0.0004 Zaprinast 51.4 >30 <0.05 Compound E 5 and others 0.38 0.37 1.0 Sulindac 5 andothers 50 50 1.0 sulfide Vinpocetine 1 >100 >100 EHNA 2,5 >100 3.7Indolidan 3 31 >100 <0.31 Rolipram 4 >100 >100 Sildenafil 5 .0003 >10<.00003

By contrast, sulindac sulfide and Compound E and competitively inhibitedboth peaks A and B phosphodiesterases at the same potency (IC₅₀=0.38 μMfor PDE peak A; 0.37 μM for PDE peak B).

There is significance for the treatment of neoplasia and the screeningof useful compounds for such treatment in the fact that peak B iszaprinast-insensitive whereas peaks A and B are both sensitive tosulindac sulfide and Compound E. We have tested zaprinast, E4021 andsildenafil to ascertain whether they induce apoptosis or inhibit thegrowth of neoplastic cells, and have done the same for Compound E. Asexplained below, zaprinast does not have significant apoptosis-inducingor growth-inhibiting properties, whereas sulindac sulfide and Compound Eare precisely the opposite. In other words, the ability of a compound toinhibit both PDE peaks A and B correlates with its ability to induceapoptosis in neoplastic cells, whereas if a compound (e.g., zaprinast)has specifity for PDE peak A only, that compound will not induceapoptosis.

E. Insensitivity of PDE Peak B To Incubation With cGMP-Dependent ProteinKinase G

Further differences between PDE peaks A and B were observed in theirrespective cGMP-hydrolytic activities in the presence of varyingconcentrations of cGMP-dependent protein kinase G (which phosphorylatestypical PDE5). Specifically, peak A and peak B fractions were incubatedwith different concentrations of protein kinase G at 30° C. for 30minutes. Cyclic GMP hydrolysis of both peaks has assayed afterphosphorylation was attempted. Consistent with previously publishedinformation about PDE5, Peak A showed increasing cGMP hydrolysisactivity in response to protein kinase G incubation, indicating thatPeak A was phosphorlyated. Peak B was unchanged, however (i.e. was notphosphorylated and insensitive to incubation with cGMP-dependent proteinkinase G). These data are consistent with Peak A being a PDE5 familyisoform and Peak B being a novel cGMP PDE.

II. Screening Pharmaceutical Compositions Using The New PDE

A. In General

The novel PDE of this invention is useful to identify compounds that canbe used to treat or prevent neoplasms, and that are not characterized byserious side effects.

Cancer and precancer may be thought of as diseases that involveunregulated cell growth. Cell growth involves a number of differentfactors. One factor is how rapidly cells proliferate, and anotherinvolves how rapidly cells die. Cells can die either by necrosis orapoptosis depending on the type of environmental stimuli. Celldifferentiation is yet another factor that influences tumor growthkinetics. Resolving which of the many aspects of cell growth is affectedby a test compound is important to the discovery of a relevant targetfor pharmaceutical therapy. Screening assays based on this technologycan be combined with other tests to determine which compounds havegrowth inhibiting and pro-apoptotic activity.

This invention is the product of several important discoveries. First,the present inventors discovered that desirable inhibitors of tumor cellgrowth induce premature death of cancer cells by apoptosis (see, Piazza,G. A., et al., Cancer Research, 55(14), 3110-16, 1995). Second, thepresent inventors unexpectedly discovered compounds that selectivelyinduce apoptosis without substantial COX inhibition also inhibit PDE5.In particular, and contrary to leading scientific studies, desirablecompounds for treating ncoplastic lesions inhibit PDE5 (EC 3.1.4.17).PDE5 is one of at least ten genc families of phosphodiesterase. PDE5 andthe novel PDE of this invention are unique in that they selectivelydegrade cyclic GMP and not cAMP, while the other famlies of PDEselectively degrade/hydrolyze cAMP and not cGMP or non-selectivelydegrade both cGMP and cAMP. Preferably, desirable compounds used totreat neoplasia do not substantially inhibit non-selective or cAMPdegrading phosphodiesterase types.

B. COX Screening

A preferred embodiment of the present invention involves determining thecyclooxygenase inhibition activity of a given compound, and determiningthe cGMP specific PDE inhibitory activity of the compound. The testcompounds are scored for their probable ability to treat neoplasticlesions either directly or indirectly by comparing their activitiesagainst known compounds useful for treating neoplastic lesions. Astandard compound that is known to be effective for treating neoplasticlesions without causing gastric irritation is5-fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetic acid(“exisulind”). Other useful compounds for comparative purposes includethose that are known to inhibit COX, such as indomethacin and thesulfide metabolite of sulindac:5-fluoro-2-methyl-1-(p-methylsulfinylbenzylidene)-3-indenylacetic acid(“sulindac sulfide”). Other useful compounds for comparative purposesinclude those that are known to inhibit (cGMP-specific PDEs, such as1-(3-chloroanilino)-4-phenyphthalazine (“MY5445”).

As used herein, the term “precancerous lesion” includes syndromesrepresented by abnormal neoplastic, including dysplastic, changes oftissue. Examples include dysplastic growths in colonic, breast, prostateor lung tissues, or conditions such as dysplastic nevus syndrome, aprecursor to malignant melanoma of the skin. Examples also include, inaddition to dysplastic nevus syndromes, polyposis syndromes, colonicpolyps, precancerous lesions of the cervix (i.e., cervical dysplasia),esophagus, lung, prostatic dysplasia, prostatic intraneoplasia, breastand/or skin and related conditions (e.g., actinic keraosis), whether thelesions are clinically identifiable or not.

As used herein, the terms “carcinoma” or “cancer” refers to lesionswhich are cancerous. Examples include malignant melanomas, breastcancer, prostate cancer and colon cancer. As used herein, the terms“neoplasia” and “neoplasms” refer to both cancerous and pre-cancerouslesions.

As used herein, the abbreviation PG represents prostaglandin; PSrepresents prostaglandin synthetase; PGE₂ represents prostaglandin E₂;PDE represents phosphodiesterase; COX represents cyclooxygenase; cyclicnucleotide, RIA represents - radioimmunoassay.

COX inhibition by a test compound can be determined by either of twomethods. One method involves measuring PGE₂ secretion by intact HL-60cells following exposure to the compound being screened. The othermethod involves measuring the activity of purified cyclooxygenases(COXs) in the presence of the compound. Both methods involve protocolspreviously described in the literature, but preferred protocols are setforth below.

Compounds of can be evaluated to determine whether they inhibit theproduction of prostaglandin E₂ (“PGE₂”), by measuring PGE₂. Using anenzyme immunoassay (EIA) kit for PGE₂, such as commercially availablefrom Amersham, Arlington Heights, Ill. U.S.A. Suitable cells includethose that make an abundance of PG, such as HL-60 cells. HL-60 cells arehuman promyelocytes that are differentiated with DMSO into maturegranulocytes (See, Collins, S. J., Ruscetti, F. W., Gallagher, R. E. andGallo, R. C., “Normal Functional Characteristics of Cultured HumanPromyelocytic Leukemia Cells (HL-60) After Induction of DifferentiationBy Dimethylsulfoxide”, J. Exp. Med., 149:969-974, 1979). Thesedifferentiated cells produce PGE₂ after stimulation with a calciumionophore, A23187 (see, Kargman, S., Prasit, P. and Evans, J. F.,“Translocation of HL-60 Cell 5-Lipoxygenase”, J. Biol. Chem., 266:23745-23752, 1991). HL-60 are available from the American Type CultureCollection (ATCC:CCL240). They can be grown in a RPMI 1640 mediumsupplemented with 20% heat-inactivated fetal bovine serum, 50 μU/mlpenicillin and 50 μg/ml streptomycin in an atmosphere of 5% CO₂ at 37°C. To induce myeloid differentiation, cells are exposed to 1.3% DMSO for9 days and then washed and resuspended in Dulbecco's phosphate-bufferedsaline at a concentration of 3×10⁶ cells/ml.

The differentiated HL-60 cells (3×10⁶ cells/ml) are incubated for 15minutes at 37° C. in the presence of the compounds tested at the desiredconcentration. Cells are then stimulated by A23187 (5×10⁻⁶ M) for 15minutes. PGE₂ secreted into the external medium is measured as describedabove.

As indicated above, a second method to assess COX inhibition of a testcompound is to measure the COX activity in the presence of a testcompound. Two different forms of cyclooxygenase (COX-I and COX-2) havebeen reported in the literature to regulate prostaglandin synthesis.COX-2 represents the inducible form of COX while COX-I represents aconstitutive form. COX-I activity can be measured using the methoddescribed by Mitchell et al. (“Selectivity of NonsteroidalAnti-inflammatory Drugs as Inhibitors of Constitutive and InducibleCyclooxygenase,” Proc. Natl. Acad. Sci. USA., 90:11693-11697, 1993,which is incorporated herein by reference) using COX-I purified from ramseminal vesicles as described by Boopathy & Balasubramanian,“Purification And Characterization Of Sheep Platelet Cyclooxygenase”(Biochem. J., 239:371-377, 1988, which is incorporated herein byreference). COX-2 activity can be measured using COX-2 purified fromsheep placenta as described by Mitchell et al., 1993, supra.

The cyclooxygenase inhibitory activity of a drug can be determined bymethods known in the art. For example, Boopathy & Balasubramanian, 1988,supra, described a procedure in which prostaglandin H synthase 1 (CaymanChemical, Ann Arbor, Mich.) is incubated at 37° C. for 20 minutes with100 μM arachidonic acid (Sigma Chemical Co.), cofactors (such as 1.0 mMglutathione, 1.0 mM hydroquinone, 0.625 μM hemoglobin and 1.25 mM CaCl₂in 100 mM Tris-HCl, pH 7.4) and the drug to be tested. Followingincubation, the reaction can be terminated with trichloroacetic acid.After stopping the reaction by adding thiobarbituric acid andmalonaldehyde, enzymatic activity can then be measuredspectrophotometrically at 530 nm.

Obviously, a compound that exhibits minimal COX-I or COX-2 inhibitoryactivity in relation to its greater PDE5/novel PDE inhibitory activitymay be a desirable test compound.

The amount of COX inhibition is determined by comparing the activity ofthe cyclooxygenase in the presence and absence of the test compound.Residual (i.e., less than about 25%) or no COX inhibitory activity at aconcentration of about 100 μM is indicative that the compound should beevaluated further for usefulness for treating neoplasia. Preferably, theIC₅₀ should be greater than 1000 μM for the compound to be furtherconsidered potential use.

C. Determining Phosphodiesterase Inhibition Activity

Compounds can be screened for inhibitory effect on the activity of thenovel phosphodiesterase of this invention using either the enzymeisolated as described above, a recombinant version, or using the novelPDE together with PDE5. Alternatively, cyclic nucleotide levels in wholecells are measured by RIA and compared to untreated andzaprinast-treated cells.

Phosphodiesterase activity can be determined using methods known in theart, such as a method using radioactive ³H cyclic GMP (cGMP)(cyclic3′,5′-guanosine monophosphate) as the substrate for the PDE enzyme.(Thompson, W. J., Teraski, W. L., Epstein, P. M., Strada, S. J.,Advances in Cyclic Nucleotide Research, 10:69-92, 1979, which isincorporated herein by reference). In brief, a solution of definedsubstrate ³H-cGMP specific activity (0.2 μM; 100,000 cpm; containing 40mM Tris-HCl (pH 8.0), 5 mM MgCl₂ and 1 mg/ml BSA) is mixed with the drugto be tested in a total volume of 400 μl. The mixture is incubated at30° C. for 10 minutes with isolated PDE of this invention. Reactions areterminated, for example, by boiling the reaction mixture for 75 seconds.After cooling on ice, 100 μl of 0.5 mg/ml snake venom (O. Hannah venomavailable from Sigma) is added and incubated for 10 minutes at 30° C.This reaction is then terminated by the addition of an alcohol, e.g. 1ml of 100% methanol. Assay samples are applied to 1 ml Dowex 1-X8column; and washed with 1 ml of 100% methanol. The amount ofradioactivity in the breakthrough and the wash from the column iscombined and measured with a scintillation counter. The degree ofphosphodiesterase inhibition is determined by calculating the amount ofradioactivity in drug-treated reactions and comparing against a controlsample (a reaction mixture lacking the tested compound but with drugsolvent).

Alternatively, the ability of desirable compounds to inhibit thephosphodiesterase of this invention is reflected by an increase in cGMPin neoplastic cells exposed to a compound being screened. The amount ofPDE activity can be determined by assaying for the amount of cyclic GMPin the extract of treated cells using radioimmunoassay (RIA). In thisprocedure, HT-29 or SW-480 cells are plated and grown to confluency. Asindicated above, SW-480 contains both PDE5 and the novel PDE of thisinvention, so when PDE activity is evaluated in this fashion, a combinedcGMP hydrolytic activity is assayed simultaneously. The test compound isthen incubated with the cell culture at a concentration of compoundbetween about 200 μM to about 200 μM. About 24 to 48 hours thereafter,the culture media is removed from the cells, and the cells aresolubilized. The reaction is stopped by using 0.2N HCl/50% MeOH. Asample is removed for protein assay. Cyclic GMP is purified from theacid/alcohol extracts of cells using anion-exchange chromatography, suchas a Dowex column. The cGMP is dried, acetylated according to publishedprocedures, such as using acetic anhydride in triethylamine, (Steiner,A. L., Parker, C. W., Kipnis, D. M., J. Biol. Chem., 247(4):1106-13,1971, which is incorporated herein by reference). The acetylated cGMP isquantitated using radioimmunoassay procedures (Harper, J., Brooker, G.,Advances in Nucleotide Research, 10:1-33, 1979, which is incorporatedherein by reference). lodinated ligands (tyrosine metheyl ester) ofderivatized cyclic GMP are incubated with standards or unknowns in thepresence of antisera and appropriate buffers. Antiserum may be producedusing cyclic nucleotide-haptene directed techniques. The antiserum isfrom sheep injected with succinyl-cGMP-albumin conjugates and diluted1/20,000. Dose-interpolation and error analysis from standard curves areapplied as described previously (Seibert, A. F., Thompson, W. J.,Taylor, A., Wilbourn, W. H., Barnard, J. and Haynes, J., J. AppliedPhysiol., 72:389-395, 1992, which is incorporated herein by reference).

In addition, the culture media may be acidified, frozen (−70° C.) andalso analyzed for cGMP and cAMP.

In addition to observing increases in the content of cGMP in neoplasticcells caused by desirable test compounds, decreases in content of cAMPhave also been observed. It has been observed that a particularlydesirable compound (i.e. one that selectively induces apoptosis inneoplastic cells, but not substantially in normal cells) follows a timecourse consistent with cGMP-specific PDE inhibition as one initialaction resulting in an increased cGMP content within minutes.Secondarily, treatment of neoplastic cells with a desirableanti-neoplastic compound leads to decreased cAMP content within 24hours. The intracellular targets of drug actions are being studiedfurther, but current data support the concept that the initial rise incGMP content and the subsequent fall in cAMP content precede apoptosisin neoplastic cells exposed to desirable compounds.

The change in the ratio of the two cyclic nucleotides may be a moreaccurate tool for evaluating desirable cGMP-specific phosphodiesteraseinhibition activity of test compounds, rather than measuring only theabsolute value of cGMP, only cGMP-specific phosphodiesterase inhibition,or only the level of cGMP hydrolysis. In neoplastic cells not treatedwith anti-neoplastic compounds, the ratio of cGMP content/cAMP contentis in the 0.03-0.05 range (i.e., 300-500 fmol/mg protein cGMP contentover 6000-8000 fmol/mg protein cAMP content). After exposure todesirable anti-neoplastic compounds, that ratio increases several fold(preferably at least about a three-fold increase) as the result of aninitial increase in cyclic GMP and the later decrease in cyclic AMP.

Specifically, it has been observed that particularly desirable compoundsachieve an initial increase in cGMP content in treated neoplastic cellsto a level of cGMP greater than about 500 fmol/mg protein. In addition,particularly desirable compounds cause the later decrease in cAMPcontent in treated neoplastic cells to a level of cAMP less than about4000 fmol/mg protein.

To determine the content of cyclic AMP, radioimmunoassay techniquessimilar to those described above for cGMP are used. Basically, cyclicnucleotides are purified from acid/alcohol extracts of cells usinganion-exchange chromatography, dried, acetylated according to publishedprocedures and quantitated using radioimmunoassay procedures. lodinatedligands of derivatized cyclic AMP and cyclic GMP are incubated withstandards or unknowns in the presence of specific antisera andappropriate buffers.

Verification of the cyclic nucleotide content may be obtained bydetermining the turnover or accumulation of cyclic nucleotides in intactcells. To measure intact cell cAMP, ³H-adenine prelabeling is usedaccording to published procedures (Whalin M. E., R. L. Garrett Jr., W.J. Thompson, and S. J. Strada, “Correlation of cell-free brain cyclicnucleotide phosphodiesterase activities to cyclic AMP decay in intactbrain slices”, Sec. Mess. and Phos. Protein Research, 12:311-325, 1989,which is incorporated herein by reference). The procedure measures fluxof labeled ATP to cyclic AMP and can be used to estimate intact celladenylate cyclase or cyclic nucleotide phosphodiesterase activitiesdepending upon the specific protocol. Cyclic GMP accumulation was toolow to be studied with intact cell prelabeling according to publishedprocedures (Reynolds, P. E., S. J. Strada and W. J. Thompson, “CyclicGMP accumulation in pulmonary microvascular endothelial cells measuredby intact cell prelabeling,” Life Sci., 60:909-918, 1997, which isincorporated herein by reference).

The PDE inhibitory activity effect of a test compound can also bedetermined from a tissue sample. Tissue biopsies from humans or tissuesfrom anestesized animals arc collected from subjects exposed to the testcompound. Briefly, a sample of tissue is homogcnized in 500 μl of 6%TCA. A known amount of the homogenate is removed for protein analysis.The remaining homogenate is allowed to sit on ice for 20 minutes toallow for the protein to precipitate. Next, the homogenate iscentrifuged for 30 minutes at 15,000 g at 4° C. The supernatant isrecovered and the pellet recovered. The supernatant is washed four timeswith five volumes of water saturated diethyl ether. The upper etherlayer is discarded between each wash. The aqueous ether extract is driedin a speed vac. Once dried, the sample can be frozen for future use, orused immediately. The dried extract is dissolved in 500 μl of assaybuffer. The amount of cGMP-specific inhibition is determnined byassaying for the amount of cyclic nucleotides using RIA procedures asdescribed above.

The amount of inhibition is determined by comparing the activity of thenovel PDE in the presence and absence of the test compound. Inhibitionof the novel PDE activity is indicative that the compound is useful fortreating neoplasia. Significant inhibitory activity greater than that ofthe benchmark, exisulind, preferably greater than 50% at a concentrationof 10 μM or below, is indicative that a compound should be furtherevaluated for antineoplastic properties. Preferably, the IC₅₀ value forthe novel PDE inhibition should be less than 50 μM for the compound tobe further considered for potential use.

D. Determining Whether A Compound Reduces The Number Of Tumor Cells

In an alternate embodiment, the screening method of the presentinvention involves further determining whether the compound reduces thegrowth of tumor cells. Various cell lines can be used in the sampledepending on the tissue to be tested. For example, these cell linesinclude: SW-480 - colonic adenocarcinoma; HT-29—colonic adenocarcinoma,A-427—lung adenocarcinoma carcinoma; MCF-7—breast adenocarcinoma; andUACC-375—melanoma line; and DU145—prostrate carcinoma. Cytotoxicity dataobtained using these cell lines are indicative of an inhibitory effecton neoplastic lesions. These cell lines are well characterized, and areused by the United States National Cancer Institute in their screeningprogram for new anti-cancer drugs.

A compound's ability to inhibit tumor cell growth can be measured usingthe HT-29 human colon carcinoma cell line obtained from ATCC (Bethesda,MD). HT-29 cells have previously been characterized as a relevant colontumor cell culture model (Fogh, J., and Trempe, G. In: Human Tumor Cellsin Vitro, J. Fogh (eds.), Plenum Press, N.Y., pp. 115-159, 1975). HT-29cells are maintained in RPMI media supplemented with 5% fetal bovinecalf serum (Gemini Bioproducts, Inc., Carlsbad, Calif.) and 2 mmglutamine, and 1% antibiotic-antimycotic in a humidified atmosphere of95% air and 5% CO₂ at 37° C. Briefly, HT-29 cells are plated at adensity of 500 cells/well in 96 well microtiter plates and incubated for24 hours at 37° C. prior to the addition of test compound. Eachdetermination of cell number involved six replicates. After six days inculture, the cells are fixed by the addition of cold trichloroaceticacid to a final concentration of 10% and protein levels are measuredusing the sulforhodamine B (SRB) colorimetric protein stain assay aspreviously described by Skehan, P., Storeng, R., Scudiero, D., Monks,A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S.,and Boyd, M. R., “New Colorimetric Assay For Anticancer-Drug Screening,”J. Natl. Cancer Inst. 82: 1107-112, 1990, which is incorporated hereinby reference.

In addition to the SRB assay, a number of other methods are available tomeasure growth inhibition and could be substituted for the SRB assay.These methods include counting viable cells following trypan bluestaining, labeling cells capable of DNA synthesis with BrdU orradiolabeled thymidine, neutral red staining of viable cells, or MTTstaining of viable cells.

Significant tumor cell growth inhibition greater than about 50% at adose of 100 μM or below is further indicative that the compound isuseful for treating neoplastic lesions. Preferably, an IC₅₀ value isdetermined and used for comparative purposes. This value is theconcentration of drug needed to inhibit tumor cell growth by 50%relative to the control. Preferably, the IC₅₀ value should be less than100 μM for the compound to be considered further for potential use fortreating neoplastic lesions.

E. Determining Whether A Compound Induces Apoptosis

In a second alternate embodiment, the screening method of the presentinvention further involves determining whether the compound inducesapoptosis in cultures of tumor cells.

Two distinct forms of cell death may be described by morphological andbiochemical criteria: necrosis and apoptosis. Necrosis is accompanied byincreased permeability of the plasma membrane; the cells swell and theplasma membrane ruptures within minutes. Apoptosis is characterized bymembrane blebbing, condensation of cytoplasm and the activation ofendogenous endonucleases.

Apoptosis occurs naturally during normal tissue turnover and duringembryonic development of organs and limbs. Apoptosis also is induced bycytotoxic T-lymphocytes and natural killer cells, by ionizing radiationand by certain chemotherapeutic drugs. Inappropriate regulation ofapoptosis is thought to play an important role in many pathologicalconditions including cancer, AIDS, or Alzheimer's disease, etc.Compounds can be screened for induction of apoptosis using cultures oftumor cells maintained under conditions as described above. Treatment ofcells with test compounds involves either pre- or post-confluentcultures and treatment for two to seven days at various concentrations.Apoptotic cells are measured in both the attached and “floating”compartments of the cultures. Both compartments are collected byremoving the supernatant, trypsinizing the attached cells, and combiningboth preparations following a centrifugation wash step (10 minutes, 2000rpm). The protocol for treating tumor cell cultures with sulindac andrelated compounds to obtain a significant amount of apoptosis has beendescribed in the literature. (See, Piazza, G. A., et al., CancerResearch, 55:3110-16, 1995, which is incorporated herein by reference).The novel features include collecting both floating and attached cells,identification of the optimal treatment times and dose range forobserving apoptosis, and identification of optimal cell cultureconditions.

Following treatment with a test compound, cultures can be assayed forapoptosis and necrosis by florescent microscopy following labeling withacridine orange and ethidium bromide. The method for measuring apoptoticcell number has previously been described by Duke & Cohen,“Morphological And Biochemical Assays Of Apoptosis,” Current ProtocolsIn Immunology, Coligan et al., cds., 3.17.1-3.17.16 (1992, which isincorporated herein by reference).

For example, floating and attached cells can be collected bytrypsinization and washed three times in PBS. Aliquots of cells can becentrifuged. The pellet can then be resuspended in media and a dyemixture containing acridine orange and ethidium bromide prepared in PBSand mixed gently. The mixture can then be placed on a microscope slideand examined for morphological features of apoptosis.

Apoptosis can also be quantified by measuring an increase in DNAfragmentation in cells which have been treated with test compounds.Commercial photometric EIA for the quantitative in vitro determinationof cytoplasmic histone-associated-DNA-fragments (mono- andoligonucleosomes) are available (Cell Death Detection ELISA^(okys), Cat.No. 1,774,425, Boehringer Mannheim). The Boehringer Mannheim assay isbased on a sandwich-enzyme-immunoassay principle using mouse monoclonalantibodies directed against DNA and histones, respectively. This allowsthe specific determination of mono- and oligonucleosomes in thecytoplasmatic fraction of cell lysates.

According to the vendor, apoptosis is measured in the following fashion.The sample (cell-lysate) is placed into a streptavidin-coated microtiterplate (“MTP”). Subsequently, a mixture of anti-histone-biotin andanti-DNA peroxidase conjugate are added and incubated for two hours.During the incubation period, the anti-histone antibody binds to thehistone-component of the nucleosomes and simultaneously fixes theimmunocomplex to the streptavidin-coated MTP via its biotinylation.Additionally, the anti-DNA peroxidase antibody reacts with the DNAcomponent of the nucleosomes. After removal of unbound antibodies by awashing step, the amount of nucleosomes is quantified by the peroxidaseretained in the immunocomplex. Peroxidase is determined photometricallywith ABTS7 (2,2′-Azido-[3-ethylbenzthiazolin-sulfonate]) as substrate.

For example, SW-480 colon adenocarcinoma cells are plated in a 96-wellMTP at a density of 10,000 cells per well. Cells are then treated withtest compound, and allowed to incubate for 48 hours at 37° C. After theincubation, the MTP is centrifuged and the supernatant is removed. Thecell pellet in each well is then resuspended in lysis buffer for 30minutes. The lysates are then centrifuged and aliquots of thesupernatant (i.e. cytoplasmic fraction) are transferred into astreptavidin-coated MTP. Care is taken not to shake the lysed pellets(i.e. cell nucleii containing high molecular weight, unfragmented DNA)in the MTP. Samples are then analyzed.

Fold stimulation (FS=OD_(max)/OD_(veh)), an indicator of apoptoticresponse, is determined for each compound tested at a givenconcentration. EC₅₀ values may also be determined by evaluating a seriesof concentrations of the test compound.

Statistically significant increases of apoptosis (i.e., greater than 2fold stimulation at a concentration of 100 μM) are further indicativethat the compound is useful for treating neoplastic lesions. Preferably,the EC₅₀ value for apoptotic activity should be less than 100 μM for thecompound to be further considered for potential use for treatingneoplastic lesions. EC₅₀ is herein defined as the concentration thatcauses 50% induction of apoptosis relative to vehicle treatment.

F. Mammary Gland Organ Culture Model Tests

Test compounds identified by the above methods can be tested forantineoplastic activity by their ability to inhibit the incidence ofpreneoplastic lesions in a mammary gland organ culture system. Thismouse mammary gland organ culture technique has been successfully usedby other investigators to study the effects of known antineoplasticagents such as NSAIDs, retinoids, tamoxifen, selenium, and certainnatural products, and is useful for validation of the screening methodof the present invention.

For example, female BALB/c mice can be treated with a combination ofestradiol and progesterone daily, in order to prime the glands to beresponsive to hormones in vitro. The animals are sacrificed and thoracicmammary glands are excised aseptically and incubated for ten days ingrowth media supplemented with insulin, prolactin, hydrocortisone, andaldosterone. DMBA (7,12-dimethylbenz(a)anthracene) is added to medium toinduce the formation of premalignant lesions. Fully developed glands arethen deprived of prolactin, hydrocortisone, and aldosterone, resultingin the regression of the glands but not the premalignant lesions.

The test compound is dissolved in DMSO and added to the culture mediafor the duration of the culture period. At the end of the cultureperiod, the glands are fixed in 10% formalin, stained with alum carmine,and mounted on glass slides. The incidence of forming mammary lesions isthe ratio of the glands with mammary lesions to glands without lesions.The incidence of mammary lesions in test compound treated glands iscompared with that of the untreated glands.

The extent of the area occupied by the mammary lesions can bequantitated by projecting an image of the gland onto a digitation pad.The area covered by the gland is traced on the pad and considered as100% of the area. The space covered by each of the unregressedstructures is also outlined on the digitization pad and quantitated bythe computer.

EXPERIMENTAL RESULTS

A number of compounds were examined in the various protocols andscreened for potential use in treating neoplasia. The results of thesetests are reported below. The test compounds are hereinafter designatedby a letter code that corresponds to the following:

A—rac-threo-(E)-1-(N,N′-diethylaminoethanethio)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;

B—(Z)-5-Fluoro-2-methyl-1-(3 ,4,5-trimethoxybenzylidene)-3-acetic acid;

C—(Z)-5-Fluoro-2-methyl-1-(p-chlorobenzylidene)-3-acetic acid;

D—rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-N-acetylcysteine;

E—(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidcne)-3-indenylacetamide,N-benzyl;

F—(Z)-5-Fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetamide,N,N′-dicyclohexyl;

G ribo-(E)-1-Triazolo-[2′,3′:1″,3″]-1-(butan-1,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;and

H—rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-glutathione).

EXAMPLE 1 COX Inhibition Assay

Reference compounds and test compounds were analyzed for their COXinhibitory activity in accordance with the protocol for the COX assay,supra. FIG. 4 shows the effect of various concentrations of eithersulindac sulfide or exisulind on purified cyclooxygenase (Type 1)activity. Cyclooxygenase activity was determined using purifiedcyclooxygenase from ram seminal vesicles as described previously(Mitchell et al, supra). The IC₅₀ value for sulindac sulfide wascalculated to be approximately 1.76 μM, while that for exisulind wasgreater than 10,000 μM. These data show that sulindac sulfide, but notexisulind, is a COX-I inhibitor. Similar data were obtained for theCOX-2 isoenzyme (Thompson, et al., Journal of the National CancerInstitute, 87: 1259-1260, 1995).

FIG. 5 shows the effect of test compounds B and E on COX inhibition. COXactivity was determined as for the compounds shown in FIG. 4. The datashow that neither test compound B and E significantly inhibit COX-I.

TABLE 2 Cyclooxygenase inhibitory activity among a series of compounds %Inhibition at 100 μM Reference compounds Indomethacin 95 MY5445 94Sulindac sulfide 97 Exisulind <25   Test compounds A <25 B <25 C   87 D<25 E <25

In accordance with the protocol, spra, compounds A through E wereevaluated for COX inhibitory activity as reported in Table 2 above.Compound C was found to inhibit COX greater than 25% at a 100 μM dose,and therefore, would not be selected for further screening.

EXAMPLE 2 cGMP PDE inhibition assay

Reference compounds and test compounds were analyzed for their cGMP PDEinhibitory activity in accordance with the protocol for the assaydescribed supra. FIG. 6 shows the effect of various concentrations ofsulindac sulfide and exisulind on either PDE4 or cGMP PDE activitypurified from human colon HT-29 cultured tumor cells, as describedpreviously (W. J. Thompson et al., supra). The IC₅₀ value of sulindacsulfide for inhibition of PDE4 was 41 μM, and for inhibition of cGMP PDEwas 17 μM. The IC₅₀ value of exisulind for inhibition of PDE4 was 181μM, and for inhibition of cGMP PDE was 56 μM. These data show that bothsulindac sulfide and exisulind inhibit phosphodiesterase activity. Bothcompounds show selectivity for the cGMP PDE isoenzyme forms over PDE4isoforms.

FIG. 7 shows the effects of sulindac sulfide on either cGMP or cAMPproduction as determined in cultured HT-29 cells in accordance with theassay described, supra. HT-29 cells were treated with sulindac sulfidefor 30 minutes and cGMP or cAMP was measured by conventionalradioimmunoassay method. As indicated, sulindac sulfide increased thelevels of cGMP by greater than 50% with an EC₅₀ value of 7.3 μM (7Atop). Levels of cAMP were unaffected by treatment, although a known PDE4inhibitor, rolipram, increased cAMP (7B bottom). The data demonstratethe pharmacological significance of inhibiting cGMP PDE, relative toPDE4.

FIG. 8 shows the effect of the indicated dose of test compound B oneither cGMP PDE or PDE4 isozymes of phosphodiesterase. The calculatedIC₅₀ value was 18 μM for cGMP PDE and was 58 μM for PDE4.

FIG. 9 shows the effect of the indicated dose of test compound E oneither PDE4 or cGMP PDE. The calculated IC₅₀ value was 0.08 μM for cGMPPDE and greater than 25 μM for PDE4.

TABLE 3 cGMP PDE inhibitory activity among a series of compounds %Inhibition at 10 μM Reference compounds Indomethacin 34 MY5445 86Sulindac sulfide 97 Exisulind 39 Test compounds A <25   B <25   C <25  D 36 E 75

The above compounds in Table 3 were evaluated for PDE inhibitoryactivity, as described in the protocol supra. Of the compounds that didnot inhibit COX, only compound E was found to cause greater than 50%inhibition at 10 μM. As noted in FIG. 8, compound B showed inhibition ofgreater than 50% at a dose of 20 μM. Therefore, depending on the dosagelevel used in a single dose test, some compounds may be screened outthat otherwise may be active at slightly higher dosages. The dosage usedis subjective and may be lowered after active compounds are found atcertain levels to identify even more potent compounds.

EXAMPLE 3 Apoptosis assay

Reference compounds and test compounds were analyzed for their novel PDEinhibitory activity in accordance with the protocols for the assay,supra. In accordance with thos protocols, FIG. 10 shows the effects ofsulindac sulfide and exisulind on apoptotic and necrotic cell death.HT-29 cells were treated for six days with the indicated dose of eithersulindac sulfide or exisulind. Apoptotic and necrotic cell death wasdetermined previously (Duke and Cohen, In: Current Protocols inImmunology, 3.17.1-3.17.16, New York, John Wiley and Sons, 1992). Thedata shows that both sulindac sulfide and exisulind are capable ofcausing apoptotic cell death without inducing necrosis. All data werecollected from the same experiment.

FIG. 11 shows the effect of sulindac sulfide and exisulind on tumorgrowth inhibition and apoptosis induction as determined by DNAfragmentation. Top figure (11A); growth inhibition (open symbols, leftaxis) and DNA fragmentation (closed symbols, right axis) by exisulind.Bottom figure (11B); growth inhibition (open symbols) and DNAfragmentation (closed symbols) by sulindac sulfide. Growth inhibitionwas determined by the SRB assay after six days of treatment. DNAfragmentation was determined after 48 hours of treatment. All data wascollected from the same experiment.

FIG. 12 shows the apoptosis inducing properties of compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound E for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was 0.05 μM.

FIG. 13 shows the apoptosis inducing properties of compound B. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound B for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was approximately 175 μM.

TABLE 4 Apoptosis inducing activity among a series of compounds Foldinduction at 100 μM Reference compounds Indomethacin <2.0 MY5445   4.7Sulindac sulfide   7.9 Exisulind <2.0 E4021 <2.0 Zaprinast <2.0Sildenafil <2.0 EHNA <2.0 Test compounds A <2.0   B 3.4 C 5.6 D <2.0   E4.6

In accordance with the fold induction protocol, supra, the compounds Athrough E were tested for apoptosis inducing activity, as reported inTable 4 above. Compounds B, C and E showed significant apoptoticinducing activity, greater than 2.0 fold, at a dosage of 100 μM. Ofthese three compounds, at this dosage only B and E did not inhibit COXbut did inhibit cGMP-specific PDE.

The apoptosis inducing activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 5 below. HT-29cell were treated for 6 days with various inhibitors ofphosphodiesterase. Apoptosis and necrosis were determinedmorphologically after acridine orange and ethidium bromide labelling inaccordance with the assay described, supra. The data show that the novelcGMP-specific PDE is useful for screening compounds that induceapoptosis of HT-29 cells.

TABLE 5 Apoptosis-Induction Data for PDE Inhibitors Inhibitor ReportedSelectivity % Apoptosis % Necrosis Vehicle  8 6 8-methoxy-IBMX PDE1  2 1Milrinone PDE3 18 0 RO-20-1724 PDE4 11 2 MY5445 PDE5 80 5 IBMXNon-selective  4 13 

EXAMPLE 4 Growth inhibition assay

Reference compounds and test compounds were analyzed for their PDE5inhibitory activity in accordance with the protocol for the assay supra.FIG. 14 shows the inhibitory effect of various concentrations ofsulindac sulfide and exisulind on the growth of HT-29 cells. HT-29 cellswere treated for six days with various doses of exisulind (triangles) orsulindac sulfide (squares) as indicated. Cell number was measured by asulforhodamine assay as previously described (Piazza et al., CancerResearch, 55: 3110-3116, 1995). The IC₅₀ value for sulindac sulfide wasapproximately 45 μM and 200 μM for the exisulind. The data show thatboth sulindac sulfide and exisulind are capable of inhibiting tumor cellgrowth.

FIG. 15 shows the growth inhibitory and apoptosis-inducing activity ofsulindac sulfide. A time course experiment is shown involving HT-29cells treated with either vehicle, 0.1% DMSO (open symbols) or sulindacsulfide, 120 μM (closed symbols). Growth inhibition (15A top) wasmeasured by counting viable cells after trypan blue staining. Apoptosis(15B bottom) was measured by morphological determination followingstaining with acridine orange and ethidium bromide as describedpreviously (Duke and Cohen, In: Current Protocols in Immunology,3.17.1-3.17.16, New York, John Wiley and Sons, 1992). The datademonstrate that sulindac sulfide is capable of inhibiting tumor cellgrowth and that the effect is accompanied by an increase in apoptosis.All data were collected from the same experiment.

FIG. 16 shows the growth inhibitory activity of test compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof comound E for six days and cell number was determined by the SRBassay. The calculated IC₅₀ value was 0.04 μM.

TABLE 6 Growth inhibitory activity among a series of compounds %Inhibition at 100 μM Reference compounds Indomethacin 75 MY5445 88Sulindac sulfide 88 Exisulind <50   E4021 <50   sildenafil <50  zaprinast <50   Test compounds A 68 B 77 C 80 D 78 E 62

In accordance with the screening protocol of section supra, compounds Athrough E were tested for growth inhibitory activity, as reported inTable 6 above. All the test compounds showed activity exceeding thebenchmark exisulind at a 100 μM single dose test.

The growth inhibitory activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 7 below. HT-29cell were treated for 6 days with various inhibitors ofphospohodiesterase. Cell growth was determined by the SRB assaydescribed, supra. The data below taken with those above show thatinhibitors of the novel PDE were effective for inhibiting tumor cellgrowth.

TABLE 7 Growth Inhibitory Data for PDE Inhibitors Growth inhibitionInhibitor Reported Selectivity (IC₅₀, μM) 8-methoxy-IBMX PDE1 >200 μMMilrinone PDE3 >200 μM RO-20-1724 PDE4 >200 μM MY5445 PDE5     5 μM IBMXNon-selective >100 μM Zaprinast PDE5 >100 μM Sildenafil PDE5 >100 μME4021 PDE5 >100 μM

To show the effectiveness of this screening method on various forms ofneoplasia, compounds were tested on numerous cell lines. The effects ofsulindac sulfide and exisulind on various cell lines was determined. Thedata are shown in table 8 below. The IC₅₀ values werc determined by theSRB assay. The data show the broad effectiveness of these compounds on abroad range of neoplasias, with effectiveness at comparable dose range.Therefore, compounds identified by this invention should be useful fortreating multiple forms of neoplasia.

TABLE 8 Growth Inhibitory Data of Various Cell Lines Cell Type/ IC₅₀(μM)Tissue specificity Sulindac sulfide Exisulind Compound E* HT-29, Colon60 120 0.10 HCT116, Colon 45  90 MCF7/S, Breast 30  90 UACC375, Melanoma50 100 A-427, Lung 90 130 Bronchial Epithelial 30  90 Cells NRK, Kidney(non ras- 50 180 transformed) KNRK, Kidney (ras 60 240 transformed)Human Prostate Carci-  82 0.90 noma PC3 Colo 205 1.62 DU-145 0.10 HCT-150.60 MDA-MB-231 0.08 MDA-MB-435 0.04 *Determined by neutral red assay asdescribed by Schmid et al., in Proc. AACR Vol 39, p. 195 (1998).

EXAMPLE 5 Activity in mammary gland organ culture model

FIG. 17 shows the inhibition of premalignant lesions in mammary glandorgan culture by sulindac metabolites. Mammary gland organ cultureexperiment were performed as previously described (Mehta and Moon,Cancer Research, 46: 5832-5835, 1986). The results demonstrate thatsulindac and exisulind effectively inhibit the formation of premalignantlesions, while sulindac sulfide was inactive. The data support thehypothesis that cyclooxygenase inhibition is not necessary for theanti-neoplastic properties of desired compounds.

ANALYSIS

To identify compounds that have potential use for treating neoplasia,this invention provides a rationale for comparing experimental data oftest compounds from several protocols. Within the framework of thisinvention, test compounds can be ranked according to their potential usefor treating neoplasia in humans. Those compounds having desirableeffects may be selected for more expensive and time consuming animalstudies that are required to get approval before initiating humanclinical trials.

Qualitative data of various test compounds and the several protocols areshown in Table 9 below. The data show that exisulind, compound B andcompound E exhibit the appropriate activity to pass the screen of fourassays: lack of COX inhibition, and presence of effective cGMP-specificPDE inhibition, growth inhibition and apoptosis induction. The activityof these compounds in the mammary gland organ culture validates theeffectiveness of this invention. The qualitative valuations of thescreening protocols rank compound E best, then compound B and thenexisulind.

TABLE 9 Activity Profile of Various Compounds Mammary Gland COX PDEGrowth Organ Compound Inhibition Inhibition Inhibition Apoptosis CultureExisulind − ++ ++ ++ +++ Sulindac ++++ +++ +++ +++ − sulfide MY5445 +++++++ +++ +++ + A − − +++ ++ ++ B − +++ +++ +++ ++ D − − ++ − − E − ++++++++ ++++ ++++ F − − ++ + − G − − +++ ++ +++ H − − ++ − − Table 9 Code:Activity of compounds based on evaluating a series of experimentsinvolving tests for maximal activity and potency. −Not active +Slightlyactive ++Moderately active +++Strongly active ++++Highly active

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

We claim:
 1. A method for identifying a compound with potential fortreating neoplasia, comprising determining cyclooxygenase (COX)inhibitory activity of the compound; and determining phospodiesterase(“PDE”) inhibition activity of the compound wherein said PDE ischaracterized by (a) a greater specificity for cGMP than cAMP; (b)positive cooperative kinetic behavior in the presence of cGMP substrate;(c) submicromolar affinity for cGMP; and (d) insensitivity tophosphorylation by cGMP-dependent protein kinase wherein low COXinhibitory activity and high PDE inhibition activity identifies that thecompound has potential for treating neoplasia.
 2. The method of claim 1,further comprising determining whether the compound inhibits tumor cellgrowth in a culture; wherein inhibition of tumor cell growth is furtheridentifies that the compound has potential for treating neoplasia. 3.The method of claim 1, further comprising determining whether thecompound induces apoptosis of a tumor cell; wherein induction ofapoptosis is further identifies that the compound has potential fortreating neoplasia.
 4. The method of claim 3, further comprisingdetermining whether the compound inhibits tumor cell growth in a sample;wherein inhibition of tumor cell growth is further identifies that thecompound is useful for treating neoplasia.
 5. The method of claim 3,wherein said PDE inhibition activity is determined by: contacting thecompound with a cell culture; and determining the intracellular cyclicGMP concentration; wherein said PDE inhibitory activity greater than 50%at a concentration of 10 μM idenitifies that a compound has potentialfor treating neoplasia.
 6. The method of claim 3 further including:determining the ratio of intracellular cyclic GMP to cyclic AMP inneoplastic cells both before and after exposure of said cells to saidcompound wherein an increase in that ratio after exposure greater thanthree-fold as compared to before exposure identifies that a compound haspotential for treating neoplasia.
 7. A method of selecting a compoundfor inhibition of neoplasia, comprising determining neoplastic cellgrowth inhibitory activity of the compound; determiningphosphodiesterase (“PDE”) inhibitory activity of the compound whereinsaid PDE is characterized by (a) a greater specificity for cGMP thancAMP; (b) positive cooperative kinetic behavior in the presence of cGMPsubstrate; (c) submicromolar affinity for cGMP; and (d) insensitivity tophosphorylation by cGMP-dependent protein kinase; and selecting thecompound that exhibit neoplastic cell growth inhibitory activity andhigh PDE inhibition activity PDE as a compound to inhibit neoplasia. 8.The method of claim 7, further comprising selecting compounds where theIC₅₀ value for growth inhibitory activity is less than about 100 μM forpotential use in treating neoplasia.
 9. The method of claim 7, furthercomprising determining whether the compound induces apoptosis in a cell;and selecting compounds that induce apoptosis.
 10. The method of claim9, further comprising selecting compounds where the EC₅₀ value forapoptotic activity is less than about 100 μM.
 11. A method foridentifying compounds for tretment of neoplasia, comprising the stepsof: determining COX inhibitory activity of the compounds; determiningphosphodiesterase (“PDE”) inhibition activity of the compounds whereinsaid PDE is characterized by (a) a greater specificity for cGMP thancAMP; (b) positive cooperative kinetic behavior in the presence of cGMPsubstrate; (c) submicromolar affinity for cGMP; (d) insensitivity tophosphorylation by cGMP-dependent protein kinase; (e) insensitivity toinhibition by zaprinast; and identifying those compounds for treatingneoplasia in patients if said compounds exhibit low COX inhibitoryactivity and high PDE inhibition activity.
 12. The method of claim 11further comprising determining growth inhibitory activity of thecompounds; and identifying those compounds with phosphodiesteraseinhibitory activity substantially greater than COX inhibitory activityat concentrations exhibiting substantial growth inhibitory activity. 13.A method for screening a compound with potential for treating neoplasiacomprising determining phosphodiesterase (“PDE”) inhibition activity ofsaid compound wherein said PDE is characterized by (a) a greaterspecificity for cGMP than cAMP; (b) positive cooperative kineticbehavior in the presence of cGMP substrate; (c) submicromolar affinityfor cGMP; and (d) insensitivity to phosphorylation by cGMP-dependentprotein kinase; wherein a compound with high PDE inhibition activity haspotential for treating neoplasia.